Class II MHC (Ia) gene products play critical roles in a variety of T lymphocyte responses. A combination of immunological and molecular genetic approaches is being used to gain an understanding of the relationship between Ia structure and function. The expression of Ia molecules has been examined at the protein level following transfection of class II Alpha and Beta genes into murine L cells (fibroblasts). These studies reveal an unexpected pattern to the cell surface Ia expression obtained using different allelic Alpha and Beta genes. Genes carried on the same chromosome give more efficient expression than those on opposite chromosomes, indicating that the Alpha and Beta genes have undergone a process of co-evolution to maintain compatibility for expression. The region controlling this effect has been mapped to the first 50 amino acids of the Beta chain. Similar transfection studies using genes from different subregion (I-A vs I-E) show that allelically conserved carboxy-terminal regions of the Beta chain play no role in selective Alpha Beta chain pairing, with the same amino-terminal Beta1 segment involved in intralocus pairing again playing the predominant role. The effects of variation in Alpha Beta allelic origin, or in particular residues of the Beta chain, on antibody and T cell recognition of Ia indicate that the polymorphic regions of these chains interact to create new conformations seen by antibodies and T cell receptors. This result makes it difficult to create a physical map of the functional regions of Ia using only the mutagenesis approach, and indirect assays like T cell activation. These studies provide new insight into the evolution of histocompatibility molecules, the general processes of intracellular protein assembly and transport, and the control of T cell responses by structural variation in MHC molecules.